Aquaculture 236 (2004) 285 – 296

www.elsevier.com/locate/aqua-online

Mortality of freshwater-acclimated Litopenaeus

vannamei associated with acclimation rate,
habituation period, and ionic challenge
William J. McGraw 1, John Scarpa *
Harbor Branch Oceanographic Institution, Inc., Aquaculture Division, 5600 US 1 North,
Fort Pierce, FL 34946, USA
Received 19 November 2002; received in revised form 12 January 2004; accepted 16 January 2004

Abstract

The effect of acclimation time, habituation period, and final freshwater ion composition on the
survival of freshwater-acclimated Pacific white shrimp Litopenaeus vannamei postlarvae was
investigated. During each of three experiments, shrimp were acclimated from 30 ppt to freshwater ( < 1
ppt TDS) utilizing various acclimation times (32, 40, 48, 72 h) with a constant or variable rate of
salinity reduction. Shrimp were then held at the final acclimation salinity for 0, 1 or 2 days (habituation
period) before being transferred to challenge ion treatment solutions. Ion treatment solutions derived
from chloride-based chemicals were of the same total ion concentration, but either strongly monovalent
or strongly divalent. An acclimation time of 72 h compared to 48 h, with no habituation period,
increased shrimp survival by 27%. A 1-day habituation period compared to no habituation after 48-
h acclimation also increased mean shrimp survivals by 27%. Decreasing acclimation time at higher
salinities (i.e., 30 down to 1 ppt) and increasing acclimation time at lower salinities ( < 1 ppt) allowed
successful freshwater acclimation of shrimp within 32 h. This demonstrates a critical period for
freshwater acclimation of marine shrimp at lower salinities. The longer freshwater acclimation time
and habituation period probably allowed shrimp to equalize hemolymph ions before transfer to
ionically unbalanced challenge solutions. The survival of freshwater-acclimated shrimp can be
improved by either extending the acclimation time from 48 to 72 h or providing a habituation period of
2 days after 48-h acclimation. However, growth at these ion concentrations still needs to be verified.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Litopenaeus vannamei; Acclimation rate; Habituation period

* Corresponding author. Tel.: +1-772-465-2400; fax: +1-772-460-1857.

E-mail addresses: wjm.toto@bigpond.com (W.J. McGraw), jscarpa@hboi.edu (J. Scarpa).
1
Present address: Taste of the Ocean Pty Ltd., PO BOX 852, Sydney NSW 1230, Australia.

0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2004.01.037
286 W.J. McGraw, J. Scarpa / Aquaculture 236 (2004) 285–296

1. Introduction

Marine shrimp culture located miles away from the ocean using saline or fresh well
water is a new and fast growing sector of aquaculture in the United States (Ednoff, 2001).
The emergence of inland shrimp farming can be traced to several factors, e.g., high cost of
coastal land, environmental concerns, and the increasing demand for shrimp in the US
(Hopkins et al., 1996). Pacific white shrimp Litopenaeus vannamei have been grown
successfully in freshwater (650 ppm TDS) at Harbor Branch Oceanographic Institution
(Van Wyk et al., 1999) and in low salinity water in other areas of the US (Samocha et al.,
1998; Ednoff, 2001; Samocha et al., 2002).
Shrimp farmers using inland well water ( < 5 ppt) have experienced increased survivals
when shrimp are given a period of time in final acclimation water before transfer to ponds
(Teichert-Coddington, Green Prairie Aquafarm, personal communication, 2000). The
authors have observed acute mortality of freshwater-acclimated Pacific white shrimp L.
vannamei postlarvae when transferring shrimp from acclimation tanks to various ion
treatment solutions of the same or slightly different salinity. Shrimp would experience
immediate, or within 24 h, loss of equilibrium and mortality. This mortality was thought to
be caused by a faster than normal acclimation rate (>50% reduction in salinity per
8-h period; Van Wyk et al., 1999), an environmental ionic imbalance into which shrimp
were transferred (McGraw and Scarpa, 2003), or immediate transfer of shrimp from
acclimation solutions without a period of habituation.
Information concerning the necessary minimum ion requirements in freshwater ( < 1000
ppm TDS) for marine shrimp culture and acceptable freshwater acclimation procedures is
limited (Van Wyk et al., 1999; Anonymous, 2002; McGraw et al., 2002; McGraw and
Scarpa, 2003). The ionic composition of well water varies greatly between locations and
depth of wells (Feth, 1970). Therefore, understanding factors, such as salinity acclimation,
that affect the survival of marine shrimp in freshwater are imperative to the advancement
of the U.S. shrimp farming industry. The following experiments were designed to examine
acclimation time of marine shrimp to freshwater, time spent in final acclimation solution
before transfer (habituation period) to final solutions, and ionic composition of final
waters, to understand how these factors affect initial survival.

2. Materials and methods

Approximately 5000 specific pathogen free (SPF) 9-day-old postlarvae (PL-9) Pacific
white shrimp L. vannamei were received from an instate hatchery (Shrimp Improvement
Systems, Islamorada, FL) on three separate dates for each of three experiments. Shrimp
were maintained in 80-l aquariums with seawater (30 ppt) and fed a prepared diet (44%
protein, Bonnie, Laramore and Hopkins, Ft. Pierce, FL) ad libitum three times per day
until the age of PL-15 when acclimation to HBOI freshwater (280 ppm Cl or 7.9 mM)
began. The general experimental design consisted of four different acclimation times (32,
40, 48 and 72 h) and 0, 1 or 2 days that shrimp spent at the final salinity (habituation
period) before being transferred to one of four different experimental treatment solutions
(strongly monovalent, strongly divalent, seawater, and HBOI freshwater). Habituation
 W.J. McGraw, J. Scarpa / Aquaculture 236 (2004) 285–296 287

described here refers to a period of time when salinity remains constant while shrimp are
kept in acclimation water before being transferred to final solutions. Final treatment
solutions of different total and individual ion concentrations were included to challenge
shrimp exposed to the different acclimation times and habituation periods in order to
understand how these factors contribute to shrimp mortality. The treatment solution of
HBOI fresh water (same as acclimation water) was used as a control, while those with
different individual and total ion compositions were included to represent the extremes that
might be encountered from saline well water sources located in various areas and at
different depths.
Shrimp were acclimated to HBOI freshwater (280 ppm Cl ) at different rates using
HBOI freshwater, which was also used as the final habituation solution. This was
accomplished by placing approximately 1000 postlarvae in each of three 40-l aquaria
containing 20 l of seawater (30 ppt) and adding HBOI freshwater, according to the
experimental protocol, using an airline valve and tubing through siphon action. The
volume of water in aquaria was reduced as necessary to maintain a constant rate of
salinity reduction based on treatment design. Salinity was checked every hour with a
refractometer and HBOI freshwater addition was adjusted as necessary to maintain the
acclimation rate. When total ion concentrations in acclimation water decreased to < 1
ppt, chloride was measured using Hach DR/3 spectrophotometric methods (Hach,
Loveland, CO) and used to indicate total ion concentration in order to maintain
acclimation rate. Shrimp were fed ad libitum during acclimation, but not during the
24-h period in final treatment solutions. Temperature of acclimation water and
treatment solutions were maintained between 26 and 27 jC throughout all experiments
as temperature fluctuation is known to affect the success of acclimation of shrimp to
freshwater (Tsuzuki et al., 2000).
After acclimation to HBOI freshwater, 10 shrimp from each acclimation treatment tank
were placed in triplicate 4-l plastic containers, each containing 2 l of aerated treatment
solution, either immediately (0-day habituation period), after 1 day, or after 2 days spent in
HBOI freshwater. After 24 h, shrimp survival was checked using a probe response. All
shrimp were then removed from treatment solutions and replaced with shrimp from the
next habituation period (i.e., 0-day habituated shrimp replaced with 1-day habituated
shrimp, etc.).
Final treatment waters were one of four solutions (Table 1): strongly monovalent
(500 ppm TDS), strongly divalent (500 ppm TDS), full-strength seawater (30 ppt
TDS), or HBOI freshwater (same as final habituation solution, 546 ppm TDS).

Table 1
Concentrations of individual ions [ppm (mM)] for final treatment solutions
Treatment solution Na+ (ppm) Ca2 + (ppm) Mg2 + (ppm) K+ (ppm) Cl (ppm) TDSa (ppm)
Strongly monovalent 143 (6.2) 8 (0.1) 26 (1.1) 6 (0.15) 317 (8.9) 500
Strongly divalent 47 (2.0) 24 (0.6) 77 (3.2) 2 (0.05) 350 (9.9) 500
HBOI freshwater 181 (7.8) 44 (1.1) 31 (1.3) 10 (0.25) 280 (7.9) 546
Seawater 10,770 414 1,300 399 19,350 32,233
a
TDS = total dissolved solids, but does not include bicarbonate ion present in ion solutions, and other ion
components found in seawater and HBOI freshwater.
288 W.J. McGraw, J. Scarpa / Aquaculture 236 (2004) 285–296

Monovalent and divalent ion concentrations were derived from previous ion experi-
ments (McGraw and Scarpa, 2002, 2003) that were designed to examine survivability
and growth of shrimp in ionic solutions skewed toward the essential monovalent (Na+,
K+) or divalent (Ca2 +, Mg2 +) ions involved in shrimp osmoregulation (Schmidt-
Nielsen, 1990).
Monovalent and divalent treatment waters were created from two stock solutions: a
monovalent solution containing Na+, K+, and Cl and a divalent solution containing
Ca2 +, Mg2 +, and Cl . Divalent ions (Ca2 +, Mg2 +) and monovalent ions (Na+, K+)
were added in an approximate ratio of seawater (g of ion per kg of seawater:
Cl = 19.35, Na+ = 10.77, Mg2 + = 1.30, Ca2 + = 0.414, K+ = 0.399; monovalent ratio=
27Na/1K; divalent ration = 3.15Mg/1Ca). These treatment solutions were formulated
using chloride-based chemicals (NaCl, CaCl2
2H20, MgCl2
6H20 and KCl, Sigma, St.
Louis, MO) added to de-ionized water. Monovalent and divalent solutions were then
mixed together per volumetric proportion as 75% monovalent and 25% divalent (i.e.,
strongly monovalent) and vice versa for strongly divalent (Table 1). Magnesium
carbonate and sodium bicarbonate were added to monovalent and divalent solutions to
produce a total alkalinity of 50 mg/l. Mean pH for monovalent and divalent treatment
waters was 7.8.
Seawater (30 ppt) and HBOI freshwater (control) were obtained from onsite wells. All
ions in final treatment solutions, except Na+ and Cl , were measured using Hach DR/3
spectrophotometric methods (Hach). Chloride was measured using a titration kit (Lamotte,
Chestertown, MD, USA). Sodium concentrations were calculated. All measured ion
concentrations were within 5% of the listed calculated values. Mean pH for seawater
and freshwater was 7.6 and 8.2, respectively.
Three-way ANOVA and Student – Newman –Keuls (Lentner and Bishop, 1993) were
used to compare survival of shrimp in treatment solutions against acclimation time,
habituation period, and final ion treatment solution. Percentage survival data were trans-
formed (arcsine  square root) before statistical analyses using software (SuperAnova,
Abacus Concepts, Berkeley, CA). Differences were considered significant if P < 0.05.
Survival of shrimp in acclimation aquaria (during acclimation and habituation period)
was visually estimated and discussed but not used for statistical analysis.

2.1. Experiment I: 40-, 48- and 72-h acclimation rate

Shrimp were acclimated from seawater (30 ppt) to HBOI freshwater (280 ppm Cl ) at a
constant hourly reduction rate in 40, 48, and 72 h (i.e., 9.1, 7.6, and 5.2% reduction in salinity
per hour). A three-way ANOVA (acclimation time, habituation period and ionic challenge)
was used to compare treatment means of shrimp survival in ion treatment solutions.

2.2. Experiment II: 32-, 40- and 48-h acclimation rate with 8-h cessation

Shrimp were acclimated from seawater (30 ppt) to HBOI freshwater (280 ppm Cl ) in 32,
40, and 48 h. Acclimation stopped at the beginning of hour 15 for all treatments and salinities
were held constant for each treatment (5, 5.5, and 9 ppt, respectively) for an 8-h period
before acclimation resumed. Acclimation rates were maintained at 12.0%, 11.4%, and
 W.J. McGraw, J. Scarpa / Aquaculture 236 (2004) 285–296 289

8.2% reduction in salinity per hour for the first 14 h and 18.5%, 11.2%, and 9.6%
reduction per hour from hour 23 until the remaining time left in the 32-, 40-, and 48-
h acclimation treatment periods, respectively. Cessation described here refers to a pause
in salinity change before continuation of acclimation, while habituation (described
above) refers to a period of time when salinity remains constant and shrimp are kept
in acclimation water before being transferred to ion treatment solutions. A three-way
ANOVA was used to compare treatment means of shrimp survival in ion treatment
solutions.

2.3. Experiment III: 32- and 40-h variable acclimation rate and 48-h constant acclimation
rate

Shrimp were acclimated from seawater (30 ppt) to freshwater (350 – 380 ppm or 10 –10.7
mM Cl ) at a constant rate during the first 24 h for two acclimation treatments (32 and 40 h).
This resulted in a salinity reduction rate of 13.5% per hour (0.9 ppt TDS at the end of hour
24). The salinities for these two treatments were then reduced to HBOI freshwater (280 ppm
Cl ) in 8 or 16 h, which was associated with a reduction in salinity of 4.3% and 2.2% per
hour. The total time for these acclimation treatments was 32 and 40 h. A standard 48-
h acclimation with a constant rate of salinity reduction (50% reduction in salinity per 8 h)
was also used as a single treatment for comparison. A three-way ANOVA was used to
compare treatment means of shrimp survival in ion treatment solutions.

3. Results

3.1. Experiment I: 40-, 48- and 72-h acclimation rate

Acclimation time and habituation period did not affect 24-h shrimp survival (76 –81%
and 73 – 82%, respectively); however, final ion treatment solution significantly affected
survival (Table 2). Strongly monovalent and HBOI freshwater treatment solutions
produced overall survivals (85 – 88%) that were significantly higher compared to seawater
and divalent treatments (70%).
A significant interaction occurred between acclimation time and habituation period. In
the 48-h acclimation time, shrimp transferred immediately (0 habituation period) into
treatment solutions had low survival (58%), whereas the 40- and 72-h acclimation times
with no habituation period exhibited survivals (76%, 85%) similar to shrimp given a 1- or
2-day habituation period.
A significant interaction also occurred between acclimation time and final treatment
solutions. For the divalent solution, the 40- and 48-h acclimation times had similar survivals
(63%, 59%) while the 72-h acclimation time showed markedly increased shrimp survival
(87%). The monovalent solution produced similar survivals for the 40- and 72-h acclimation
time (92%, 90%), while the 48-h acclimation time had less survival (82%). The seawater
treatment exhibited variable shrimp survival for all acclimation times (50 – 83%). The HBOI
freshwater treatment solution produced higher shrimp survival (93%) with the 48-h accli-
mation rate compared to the 40- and 72-h rate (84%, 78%).
290 W.J. McGraw, J. Scarpa / Aquaculture 236 (2004) 285–296

Table 2
Mean survival (% F S.E., n = 3) of PL-18 Pacific white shrimp Litopenaeus vannamei in final treatment solutions
after 40-, 48-, and 72-h acclimation times and 0-, 1-, or 2-day habituation periods
Ion treat solution Acclimation time Mean
40 h 48 h 72 h 40 h 48 h 72 h 40 h 48 h 72 h ion treat

0-day habituation 1-day habituation 2-day habituation

Strongly monovalent 90 F 10 73 F 9 90 F 6 97 F 3 87 F 3 90 F 10 90 F 6 87 F 3 90 F 6 88 F 2a
Strongly divalent 53 F 9 17 F 17 87 F 8 57 F 12 80 F 10 83 F 9 80 F 10 80 F 10 90 F 10 70 F 5b
Seawater 83 F 9 50 F 21 77 F 14 73 F 3 80 F 6 63 F 12 57 F 15 80 F 6 70 F 21 70 F 4b
Freshwater 77 F 7 93 F 3 87 F 7 77 F 9 93 F 7 77 F 12 97 F 3 93 F 7 70 F 21 85 F 3a

40 h 48 h 72 h
Mean for accl. time 78 F 3 76 F 4 81 F 3

0 day 1 day 2 day

Mean for hab. period 73 F 5 80 F 3 82 F 3
Values followed by a different superscript are significantly different at P < 0.05.

3.2. Experiment II: 32-, 40- and 48-h constant acclimation with 8-h cessation

The 32-h acclimation time with 8-h cessation produced >90% shrimp mortality during
acclimation. The few remaining shrimp were pale and lethargic and were not placed in
treatment solutions. The 40-h acclimation time with 8-h cessation produced approximately
20% mortality during acclimation, while no shrimp mortality was noted in the 48-
h acclimation aquaria. Acclimation time and habituation period did not affect final shrimp
survival (84 – 86% and 80 – 89%, respectively), however, final ion treatment solution
significantly affected survival (Table 3). Mean survival for the monovalent ion treatment
(94%) was significantly greater than the divalent or seawater treatment (81%, 79%), but not
the freshwater control (86%).
A significant interaction occurred between habituation period and ion treatment sol-
utions. The monovalent solution had similarly high survival (91 – 97%) among habituation
times (0, 1, 2 days), whereas the divalent solution showed high survival (98%) with 2-day
habituation and reduced survival (72%) at 0- and 1-day habituation. The seawater treatment
produced the same survival (75%) between the 0- and 2-day habituation periods, but
showed increased survival (88%) for 1-day habituation. The HBOI freshwater control
produced the same survival for the 1- and 2-day habituation period (88%) while 0-
day habituation exhibited 82%.

3.3. Experiment III: 32- and 40-h variable acclimation rate and 48-h constant acclimation
rate

Acclimation time, habituation period, and final ion treatment did not significantly
affect shrimp survival (Table 4). Shrimp survival varied from 84% to 91% among
 W.J. McGraw, J. Scarpa / Aquaculture 236 (2004) 285–296 291

Table 3
Mean survival (% F S.E., n = 3) of PL-18 Pacific white shrimp Litopenaeus vannamei after 40- and 48-h constant
acclimation times with 8-h cessation (C*) and 0-, 1-, or 2-day habituation periods
Ion treat solution Acclimation time Mean
40 h (C) 48 h (C) 40 h (C) 48 h (C) 40 h (C) 48 h (C) ion treat

0-day habituation 1-day habituation 2-day habituation

Strongly monovalent 100 F 0 90 F 10 93 F 7 93 F 7 97 F 3 90 F 6 94 F 2a
Strongly divalent 67 F 7 73 F 7 77 F 3 70 F 6 100 F 0 97 F 3 81 F 8b
Seawater 73 F 15 77 F 15 87 F 13 90 F 10 73 F 13 77 F 9 79 F 4b
Freshwater 73 F 9 90 F 6 97 F 3 80 F 12 100 F 0 77 F 9 86 F 6a,b

40 h (C) 48 h (C)
Mean for accl. time 86 F 7 84 F 5

0 day 1 day 2 day

Mean for hab. period 80 F 7 86 F 5 89 F 6
Values followed by a different superscript are significant at P < 0.05.
* C = 8-h cessation from hours 15 to 23 during acclimation from seawater (30 ppt) to freshwater (280 ppm Cl ).

final ion treatments. A significant interaction between acclimation time and habitu-
ation period demonstrated that as acclimation time increased from 32 to 48 h, shrimp
survival for 0- and 2-day habituation increased from 86 – 89% to 84 – 94%, respec-
tively. For 1-day habituation, survival decreased from 91% to 79% as acclimation time
increased from 32 to 40 h and then increased to almost 90% for the 48-h acclimation
time.

Table 4
Mean survival (% F S.E., n = 3) of PL-18 Pacific white shrimp Litopenaeus vannamei using a 32- and 40-
h variable (V*) and 48-h constant acclimation time, three habituation periods (0, 1, 2 days) and four final
challenge ion treatment solutions
Ion treat solutions Acclimation times Mean
ion treat
32 h (V) 40 h (V) 48 h 32 h (V) 40 h (V) 48 h 32 h (V) 40 h (V) 48 h
0-day habituation 1-day habituation 2-day habituation
Strongly monovalent 93 F 3 93 F 3 97 F 3 87 F 9 77 F 7 93 F 3 83 F 7 97 F 3 83 F 3 89 F 2
Strongly divalent 90 F 6 80 F 12 70 F 6 93 F 7 63 F 3 90 F 6 73 F 3 97 F 3 97 F 3 84 F 3
Seawater 83 F 3 87 F 9 97 F 3 93 F 7 80 F 6 93 F 3 93 F 7 73 F 9 100 F 0 89 F 2
Freshwater 80 F 12 97 F 3 93 F 7 90 F 10 97 F 3 83 F 9 87 F 9 97 F 3 97 F 3 91 F 2

32 h (V) 40 h (V) 48 h
Mean for accl. time 87 F 2 86 F 2 91 F 2

0 day 1 day 2 day

Mean for hab. period 88 F 2 87 F 2 89 F 2
Values followed by a different superscript are significant at P < 0.05.
* V = variable acclimation time for 32- and 40-h time from seawater (30 ppt) to freshwater ( < 1 ppt).
292 W.J. McGraw, J. Scarpa / Aquaculture 236 (2004) 285–296

4. Discussion

4.1. Effect of acclimation time

Van Wyk et al. (1999) suggested postlarval shrimp should be acclimated from full
strength seawater (35 ppt) to freshwater (300 ppm Cl ) at a rate of 50% reduction in
salinity per 8 h over a 48-h period. Although 48-h acclimation from seawater to
freshwater produced acceptable survival for this study, immediate transfer of shrimp
to a strongly divalent solution after freshwater acclimation showed markedly de-
creased survivals compared to transfer after 72-h acclimation. The 72-h acclimation
treatment probably allowed greater equalization of ions between shrimp hemolymph
and the surrounding acclimation medium, thereby reducing osmoregulatory stress in
the shrimp that resulted in higher survival. This balance of ions between the
environment and shrimp hemolymph provided by the extended acclimation time
(Bursey and Lane, 1971; Lin et al., 2000) probably enabled shrimp to survive ion
imbalance of divalent solutions of equal total ion concentration. This concurs with
results of Pantastico and Oliveros (1980) who found that acclimation of Peneaus
monodon (PL-20, 35, and 90) from 16 to 0 ppt conducted over a period of 3 days
significantly increased survivals of all three PL ages compared to a 1- or 2-day
acclimation period.
Acclimation times of 32 h (Experiment III) and 40 h (all exps) yielded similar
survival to 72-h acclimation time after transfer to final treatment solutions. However,
during the 32- and 40-h acclimation times, shrimp mortality was noted in the
acclimation aquaria. Shrimp mortalities that occurred in acclimation aquaria were often
consumed by cohorts, which made estimation of shrimp survival in aquaria difficult
(survival in acclimation aquaria was not used in analysis). Osmoregulatory ability of
crustaceans that inhabit environments with changing salinity varies between populations,
cohorts, seasons and temperatures (Mantel and Farmer, 1983). Osmoregulatory ability
has been suggested by Laramore et al. (2001) to be variable between and within shrimp
cohorts. Salinity shock treatment is used in shrimp growout operations as a test before
stocking shrimp into ponds to show the health status of shrimp, which have been
recently received from hatcheries (Villalon, 1991). Therefore, it seems probable that
some of the weaker shrimp within the 32- and 40-h acclimation aquaria succumbed to
salinity stress leaving the ‘‘hardier’’ individuals for transfer into ion solutions, which
showed similar survival to longer acclimation times (48 and 72 h) in all ion treatment
solutions.
An extended acclimation period at very low salinities (280 – 380 ppm Cl ) showed
an obvious advantage in increased survival of shrimp for this study. This concurs with
the study conducted by Pantastico and Oliveros (1980) who determined the critical
period of freshwater acclimation of P. monodon from 16 ppt occurred at salinities < 6
ppt with peak mortalities evident at salinities < 2 ppt. Successful freshwater acclima-
tion rates for L. vannamei suggested by Van Wyk et al. (1999) shows salinity
reduction decreased from 2 ppt/h for the first 8 h down to 0.063 ppt/h for the last 8 h
during a 48-h period, demonstrating the importance of an extended acclimation period
at low salinities.
 W.J. McGraw, J. Scarpa / Aquaculture 236 (2004) 285–296 293

4.2. Effect of habituation period

Although not a statistically significant factor, a 1-day habituation period generally

produced an increase in shrimp survival over immediate transfer to final treatment
solutions. Survival increased 9 –31% in the divalent ion solution as habituation period
increased from 0 to 2 days. Shrimp in the 48-h acclimation treatment showed an
increase in survival up to 27% for the 1- or 2-day habituation period over immediate
transfer. Ions in the hemolymph of crustaceans have been previously shown to equalize
to steady concentrations after 24 h. P. monodon transferred from 45 to 15 ppt needed 24
h until ionic and osmotic changes in the hemolymph ceased (Lin et al., 2000).
Farfantepenaeus duorarum transferred from full strength seawater to salinities ranging
from 7 to 28 ppt also needed 24 h to adjust ion concentrations to that of the changing
medium (Bursey and Lane, 1971). Two species of crayfish (Procambarus clarkii and P.
zonangulus), found in fresh and brackish water, experienced osmotic stability in
hemolymph after 48 h when transferred from freshwater to a salinity of 20 ppt (Newsom
and Davis, 1994).
Although shrimp given 2-day habituation showed an increase in survival when
transferred to treatment solutions, other studies show longer-term changes to low-salinity
environments. Shrimp kept in low-salinity water for several weeks are thought to have
increased tolerance to low-salinity ion fluctuation possibly due to long-term physiolog-
ical adaptation to hypotonic conditions. Permeability of crustacean gills to the external
medium may also be modified in response to long-term acclimation to dilute media
(Mantel and Farmer, 1983). Invertebrates collected from natural low-salinity environ-
ments were more tolerant of low-salinity test solutions while animals taken from
seawater were more tolerant of high-salinity test waters (Stancyk and Shaffer, 1977;
Kangas and Skoog, 1978).

4.3. Salinity change vs. ion change

The ionic make up of shrimp hemolymph is predominantly (88%) sodium and chloride
(Chen and Chen, 1996). Therefore, it was expected that the divalent solution would be
more challenging to shrimp and produce increased mortality compared to the monovalent
or the control solution (HBOI freshwater). Monovalent and HBOI freshwater have ion
ratios similar to shrimp hemolymph. These treatment solutions would be expected to
produce less osmotic stress in shrimp and have higher survivals than the strongly divalent
solution, which they did.
This present study also demonstrated that shrimp acclimated to freshwater can be
immediately transferred into full strength seawater with good survivals. Strongly
divalent ion treatment solutions, which had similar salinity as the final acclimation
water, appear to exhibit an equal or greater detrimental effect on shrimp survival as
compared to a change in salinity of nearly 30 ppt. L. vannamei (>PL-15) are capable
of tolerating a high rate of salinity reduction, e.g., from 24 to 1 ppt in about 5
h (McGraw et al., 2002). Fenneropenaeus indicus (PL 7– 22) can survive an abrupt
salinity change of up to 10 ppt, but transfer of shrimp from 30 to 5 and 10 ppt
resulted in mass mortalities (Kumlu and Jones, 1995). Experience of the authors have
294 W.J. McGraw, J. Scarpa / Aquaculture 236 (2004) 285–296

shown L. vannamei (PL 15– 20) can survive a salinity change from 30 to 15 ppt with
limited mortality but changes >20 ppt produced greatly increased mortality. However,
it is evident from the present study that PL-15 shrimp can survive greater hypertonic
salinity changes than hypotonic changes as shrimp transferred from freshwater to 30
ppt had good survival. This may be related to the increase in enzyme activity and
reduced gill permeability that would be prevalent from freshwater acclimation (Siebers
et al., 1982, 1983). Shrimp hemolymph is isotonic to external salinities at about 25
ppt (Castille and Lawrence, 1981; Parado-Estepa et al., 1987) so immediate transfer to
higher salinities from low-salinity acclimation water would seem to benefit shrimp in
returning hemolymph ion concentrations back to previous, less stressful levels before
acclimation.

4.4. Importance of PL age and development

Gill development in relation to osmoregulatory ability in penaeids is correlated with

PL age (PL-12 to PL-15). For the present study, all PLs used for experiments were
purposefully the same age (PL-15) before acclimation began. Smaller animals have a
larger surface to volume ratio and in general have higher uptake capacities to balance a
larger salt loss per unit weight (Mantel and Farmer, 1983) during hypotonic conditions.
Other work has shown the importance of PL age (PL 10– 20) in relation to osmoreg-
ulation ability when acclimating shrimp from 24 ppt to a salinity of 1 ppt (McGraw et
al., 2002). Shrimp at the PL15 –20 stage showed significantly higher survival when
exposed to low salinities (1 ppt) and higher rate of salinity reduction (47% per h) from
24 to 1 ppt. However, osmoregulatory ability in relation to age and individual ion
manipulation by shrimp remains unclear. Vargas-Albores and Ochoa (1992) showed
three different size groups (11.0 – 12.6, 12.7 –14.3 and 14.4 – 16.0 cm) of P. stylirostris
maintained constant Na+ concentrations in the hemolymph while K+ levels and
osmolality were significantly different between size groups. All animals were taken
from the same location. Larger animals tended to accumulate more K+ and possess
greater osmolality. Potassium and Na+ tended to increase in a constant ratio with
increasing hemolymph osmolality.

5. Conclusion

The present study demonstrated that mortality of freshwater acclimated PL-15 L.

vannamei placed in solutions of various ion concentrations and salinities can be reduced
by allowing an extended acclimation time from 48 to 72 h or by increasing time in
acclimation water (habituation period) before transfer to strongly divalent solutions of the
same salinity. Increasing habituation period did not show an increase in tolerance of
shrimp to a change of 30 ppt total salinity as direct transfer of shrimp from freshwater to
seawater resulted in high survivals. Solutions with greater monovalent compared to
divalent ions showed significantly better survival after successful freshwater acclimation.
Increasing acclimation time at lower salinities increased shrimp survival at shorter than
recommended freshwater acclimation times (i.e. 48 h).
 W.J. McGraw, J. Scarpa / Aquaculture 236 (2004) 285–296 295

Acknowledgements

The authors thank the Harbor Branch Institute post doctorate fellowship program for
providing funds for this research. Special thanks to the HBOI library personnel for
providing many valuable library searches and interlibrary loan documents. Gratitude is
also expressed to those who have critically reviewed this manuscript. This is HBOI
contribution 1527.

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